Elucidating the Role and Regulation of Proteostasis in Hepatic Fibrogenesis

阐明蛋白质稳态在肝纤维形成中的作用和调节

基本信息

批准号：
10718882
负责人：
Jessica L Maiers
金额：
$ 42.65万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-01 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10718882
关键词：
Apoptosis Autophagocytosis Binding Cell Separation Cell Survival Cells Cellular Stress Chemicals Cicatrix Cirrhosis Data Deposition Development Endoplasmic Reticulum Excision Extracellular Space Failure Fibrosis GADD45A gene Genes Genetic Transcription Genomics Goals Golgi Apparatus Health Hepatic Fibrogenesis Hepatic Stellate Cell Hepatocyte Impairment In Vitro Injury Intervention Liver Liver Cirrhosis Liver Failure Liver Fibrosis Lysosomes Mediating Mus Pathway interactions Patients Phosphotransferases Post-Translational Regulation Process Procollagen Production Protein Secretion Proteins Proteome Proteomics Regulation Research Role Secretory Cell Signal Transduction Stress Testing Therapeutic Tissues Transforming Growth Factor beta Universities Validation antifibrotic treatment arm chronic liver injury end stage liver disease endoplasmic reticulum stress fibrogenesis gene induction global health in vivo insight liver function liver injury lysosomal proteins misfolded protein new therapeutic target non-alcoholic fatty liver disease posttranscriptional protein degradation protein folding protein transport proteostasis receptor response therapeutically effective trafficking transcriptome sequencing

项目摘要

Liver cirrhosis is a major health crisis caused by chronic liver injury. Liver injury leads to fibrosis which impairs liver function and can progress to cirrhosis and end-stage liver disease if unchecked. Fibrosis can reverse with removal of the injury; however, no therapeutics effectively targeted fibrosis. The major fibrogenic cells in the liver are hepatic stellate cells (HSCs), which produce and secrete matrix proteins into the extracellular space to drive fibrogenesis. Production of matrix proteins in fibrogenic HSCs exceeds the folding capacity of the endoplasmic reticulum (ER), leading to ER stress and induction of the Unfolded Protein Response (UPR). The UPR propa- gates signaling cascades to increase protein folding demands, restore proteostasis, and promote cell survival (adaptive UPR); however prolonged ER stress leads to apoptosis. We propose that targeting the adaptive UPR in HSCs is an effective anti-fibrotic strategy, through disrupting proteostasis, promting apoptosis, and limiting fibrogenesis. A major effector of the adaptive UPR is ATF6α. We found that ATF6α is necessary and sufficient to promote HSC activation in vitro, and HSC-specific loss of ATF6α limited fibrogenesis in vivo; however, the pro-fibrotic mechanisms downstream of ATF6α are unclear. To gain insight into these mechanisms, we performed RNAseq on HSCs isolated from fibrotic Atf6aHSCΔ/Δ mice, revealing disruption of several pathways compared to Atf6afl/fl HSCs, including pathways crucial for maintaining proteostasis through autophagy, protein secretion, and cell survival. We hypothesize that ATF6α drives fibrogenesis through regulating proteosta- sis in fibrogenic HSCs. Based on RNAseq as well as proteomics data from patient derived HSCs, this proposal will focus on how ATF6α regulates proteostasis to drive fibrogenesis through three distinct mechanisms: protein degradation, regulation of pro-survival signals, and protein trafficking. Degrading misfolded proteins is crucial for relieving ER stress. Misfolded proteins in the ER can undergo ER-to-lysosomal associated degradation (ER- LAD), with ERLAD receptors targeting misfolded proteins for lysosomal degradation. ERLAD activity and ERLAD receptors increased in fibrogenic HSCs in an ATF6α-dependent manner. Aim 1 will study how ATF6α regulates ERLAD to reduce ER stress and promote HSC survival and fibrogenesis. Second, ATF6α is crucial for survival of secretory cells, and we show that ATF6α induces expression of GADD45A, a protein which binds to and regulates several kinases. We show that GADD45A loss limits HSC activation in vitro, thus Aim 2 will study how ATF6α regulates GADD45A expression and association with pro-survival kinases to promote HSC survival and fibrogenesis in vitro and in vivo. Finally, we identified a potential role for ATF6α in procollagen I secretion through SORT1. Aim 3 will utilize a combination of in vitro mechanistic studies, proteomics, and in vivo studies to reveal how ATF6α regulation of SORT1 promotes procollagen I trafficking and secretion to drive fibrogenesis. Together these studies will elucidate how ATF6α coordinates signaling mechanisms to restore proteostasis in activated HSCs, and identify novel therapeutic targets to limit liver fibrosis in vivo.

肝硬化是由慢性肝损伤引起的严重健康危机，导致肝纤维化损害。如果不加以控制，可能会发展为肝硬化和终末期肝病，纤维化可以逆转。去除损伤；然而，没有有效针对肝脏纤维化细胞的治疗方法。是肝星状细胞（HSC），它产生基质蛋白并将其分泌到细胞外空间以驱动纤维化 HSC 中基质蛋白的产生超过内质的折叠能力。网状结构 (ER)，导致 ER 应激并诱导未折叠蛋白反应 (UPR)。门信号级联增加蛋白质折叠需求、恢复蛋白质稳态并促进细胞存活（适应性 UPR）；然而，长期的 ER 应激会导致细胞凋亡。 HSC 中的 UPR 是一种有效的抗纤维化策略，通过破坏蛋白质稳态、促进细胞凋亡、适应性 UPR 的主要效应物是 ATF6α。我们发现 ATF6α 是必需的。并足以在体外促进 HSC 激活，而 HSC 特异性的 ATF6α 损失限制了体内纤维形成；然而，ATF6α 下游的促纤维化机制尚不清楚。我们对从纤维化 Atf6aHSCΔ/Δ 小鼠中分离的 HSC 进行了 RNAseq，揭示了多个途径的破坏与 Atf6afl/fl HSC 相比，包括通过自噬、蛋白质维持蛋白质稳态的关键途径我们努力发现 ATF6α 通过调节蛋白质稳态来驱动纤维形成。该提案基于 RNAseq 以及患者来源的 HSC 的蛋白质组学数据。将重点关注 ATF6α 如何通过三种不同的机制调节蛋白质稳态以驱动纤维发生：蛋白质降解、促生存信号的调节和蛋白质运输对于错误折叠的蛋白质至关重要。缓解内质网应激。内质网中错误折叠的蛋白质可能会发生内质网至溶酶体相关的降解（ER-）。 LAD），ERLAD 受体针对错误折叠蛋白进行溶酶体降解。纤维化 HSC 中受体以 ATF6α 依赖性方式增加目标 1 将研究 ATF6α 如何调节。 ERLAD 可减少 ER 应激并促进 HSC 存活和纤维形成其次，ATF6α 对于存活至关重要。分泌细胞，我们发现 ATF6α 诱导 GADD45A 的表达，GADD45A 是一种与我们发现 GADD45A 的缺失会限制 HSC 的体外激活，因此目标 2 将研究如何调节这些激酶。 ATF6α 调节 GADD45A 表达并与促生存激酶关联，以促进 HSC 存活和最后，我们确定了 ATF6α 在 I 型前胶原分泌中的潜在作用。 SORT1。目标 3 将结合体外机制研究、蛋白质组学和体内研究来揭示 ATF6α 对 SORT1 的调节如何促进 I 型前胶原的运输和分泌，从而共同驱动纤维形成。这些研究将阐明 ATF6α 如何协调信号机制以恢复蛋白质稳态激活 HSC，并确定限制体内肝纤维化的新治疗靶点。